LOCUS       sequence-248025-        3231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing sgRNA2 (TACCACATTTGTAGAGGTT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3231)
  AUTHORS   Rui Y, Varanasi M, Mendes S, Yamagata HM, Wilson DR, Green JJ
  TITLE     Poly(Beta-Amino Ester) Nanoparticles Enable Nonviral Delivery of 
            CRISPR-Cas9 Plasmids for Gene Knockout and Gene Deletion.
  JOURNAL   Mol Ther Nucleic Acids. 2020 Apr 21;20:661-672. doi: 
            10.1016/j.omtn.2020.04.005.
  PUBMED    32380416
REFERENCE   2  (bases 1 to 3231)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther 
            Nucleic Acids."; date: "2020-04-21"; volume: "20:661-672. doi"; 
            pages: " 10.1016/j.omtn.2020.04.005"
COMMENT     Sequence Label: pPTG-GG-sg2+sg3
FEATURES             Location/Qualifiers
     source          1..3231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(294..1085,1086..1154))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     917..936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1155..1259)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1391..1639
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1391..1411
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1562..1581
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1742..1817
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1915..1990
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2019..2035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2019..2035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2032..2054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2043..2059
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2067..2073,2074..2091,2092..2097))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2112..2133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2192..2387
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2238..2373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2393..2527
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2430..2449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2484..2503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2595..2612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(2766..3231,1..123))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2846..2865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
       61 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
      121 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
      181 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
      241 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
      301 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
      361 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
      421 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
      481 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
      541 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      601 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      661 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      721 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      781 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      841 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      901 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      961 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     1021 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     1081 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     1141 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     1201 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     1261 acatttcccc gaaaagtgcc acctgggtcg acattgatta ttgactagta gtggagaatt
     1321 ctgtacaaaa aagcaggctt taaaggaacc aattcagtcg actggatccg gtaccaaggt
     1381 cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     1441 tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     1501 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     1561 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     1621 gtggaaagga cgaaacacct ctagaacaaa gcaccagtgg tctagtggta gaatagtacc
     1681 ctgccacggt acagacccgg gttcgattcc cggctggtgc agaacctcta caaatgtggt
     1741 agttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     1801 tggcaccgag tcggtgcaac aaagcaccag tggtctagtg gtagaatagt accctgccac
     1861 ggtacagacc cgggttcgat tcccggctgg tgcagcaatg tatcttatca tgtcgtttta
     1921 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     1981 gagtcggtgc tttttttttt aagcttggcg taatcatggt catagctgtt tcctgtgtga
     2041 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2101 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2161 cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc
     2221 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2281 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2341 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt
     2401 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2461 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     2521 ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2581 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2641 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2701 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2761 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2821 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2881 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2941 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3001 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3061 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3121 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3181 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct g
//
